The facultative intracellular actinomycete, Rhodococcus equi, is a frequent and serious pathogen of young horses (foals) and an occasional but equally serious opportunistic pathogen of immunocompromised people [Emmons, 1991 #402]. It is one of the few bacteria capable of intramacrophage survival and replication, and thus, the study of this organism can provide insights into the biology of intracellular parasitism. The native host cell of R. equi is the alveolar macrophage, infected through inhalation of bacteria present in contaminated soil or dust. Pyogranulomatous pneumonia ensues which can be life threatening if appropriate long-term antibiotic therapy is not initiated or continued. Strains of R. equi isolated from pneumonic foals all possess a large (80.6 kb) virulence plasmid [Tkachuk-Saad, 1991 #382] [Takai, 1991 #383], the entire nucleotide sequence of which has been recently determined [Takai, 2000 #43]. This plasmid is essential for virulence, as plasmid curing attenuates the bacterium and renders it harmless to foals [Giguere, 1999 #87]. Plasmid loss yields a strain incapable of replicating intracellularly and thus, plasmid encoded factors are necessary for both intramacrophage growth and disease development in horses [Hondalus, 1994 #275][Giguere, 1999 #87]. Interestingly, the virulence plasmid is only present in a subset of R. equi strains isolated from humans [Takai, 1995 #236] with rhodococcal pneumonia, indicating that chromosomally derived gene products also influence the disease process.
At present, a fundamental understanding of most aspects of R. equi pathogenesis is lacking. Very little is known about the genetic basis for R. equi virulence, and no single gene product has been definitely identified as a determinate of virulence. There is therefore a need to establish the bacterial requirements for virulence, to understand why disease develops in some hosts and not others, to ascertain how best to treat rhodococcal disease when it does develop, and importantly, to construct preventative vaccines. In order to meet these goals, R. equi mutant strains must be created, the study of which will aid in the dissection of the molecular basis of R. equi pathogenesis.
Riboflavin (vitamin B2) is the precursor of the coenzymes flavin mononucleotide phosphate (FMN) and flavin adenine dinucleotide phosphate (FAD), compounds essential for growth and cell division. Riboflavin is synthesized by plants, fungi and bacteria but not by higher eukaryotes. Some bacteria are readily able to make use of exogenous sources of riboflavin, but a number, for example E. coli and other Enterobacteriaceae lack a transport system for riboflavin uptake and thus, are unable to efficiently utilize environmental sources of riboflavin. Even if a bacterium is in possession of an adequate uptake system, the mammalian environment may be so limiting in riboflavin availability that bacterial growth is prevented. Such a scenario was documented by studies of the veterinary pathogen Actinobacillus pleuropneumoniae, in which a riboflavin auxotroph was demonstrated to be incapable of causing disease in swine.